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Industrial & Engineering Chemistry Research
Biodegradable, In Situ-Forming Cell-Laden Hydrogel Composites of Hydroxyapatite Nanoparticles for Bone Regeneration Brendan M. Watson1, Tiffany N. Vo1, Paul S. Engel, 2 Antonios G. Mikos1,* Departments of 1Bioengineering and 2Chemistry, Rice University, Houston, TX 77030, USA
Manuscript submitted to the Special Issue of I&EC Research, Doraiswami Ramkrishna Festschrift *Corresponding Author Antonios G. Mikos Department of Bioengineering Rice University 6500 Main Street Houston, Texas 77030, USA Tel: 713-348-5355 Fax: 713-348-4244 E-mail:
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Key words: hydrogel composite, poly(N-isopropyl acrylamide), monoacryloxyethyl phosphate, hydroxyapatite nanoparticles, mesenchymal stem cell encapsulation
Abstract
Hydroxyapatite nanoparticles (HANPs) were suspended in injectable, biodegradable, phosphatecontaining, dual-gelling macromer solutions that were used to encapsulate mesenchymal stem cells (MSCs) within stable hydrogel composites when elevated to physiologic temperature. The suspension of HANPs at 0.75 %w/v within the hydrogels was found to have no significant effect on the swelling ratio or the compressive modulus. The MSCs were shown to survive the encapsulation process and live cells were detected within the hydrogel composites for up to 28 days. The activity of osteogenic marker alkaline phosphatase increased with time in cell-laden hydrogel composites over the 28 days in osteogenic medium, suggesting that the joint combination of encapsulated MSCs within an HANP- and phosphate-containing hydrogel can enhance enzyme activity to assist in hydrogel mineralization. This is observed in the calcium biochemical assays, where the incorporation of HANPs significantly improved both cell-laden and acellular hydrogel composite mineralization over time. Hydrogel nanocomposites that form in situ while facilitating cell delivery and mineralization are promising material for craniofacial bone tissue engineering.
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Introduction: Tissue engineering traditionally merges biomaterials, cells, and bioactive factors together to improve regeneration of destroyed or injured tissue. Craniofacial bone tissue engineering strategies to repair congenital abnormalities and trauma could provide great clinical benefit. Currently, autologous grafts are the primary means of craniofacial bone repair, but this strategy is limited both by donor-site morbidity and the difficulty in shaping grafts to properly match the contours of the face.1 Compared to autologous grafts, injectable biomaterials that can form in situ have the potential to more appropriately match facial contours while filling craniofacial defects, which can provide a better foundation for guiding tissue growth.2 Furthermore, injectable biomaterials also have the potential to deliver stem cells that can accelerate regeneration of tissue, making them promising candidates for craniofacial bone tissue engineering. Mesenchymal stem cells (MSCs) are often used in bone tissue engineering strategies because they have a high proliferative capacity and can be harvested more easily than osteoblasts.3 However, they require osteogenic differentiation in order to generate bone. Consequently, it is important for bone regeneration scaffolds to provide osteogenic cues to help facilitate cell-induced mineralization by encapsulated MSCs so that they can generate bone tissue to fill the defect.4 Hydroxyapatite nanoparticles (HANPs) have been shown to improve biomineralization in a wide variety of scaffold materials,5,6 and have been used specifically as bioactive factors in hydrogels to improve mineralization and osteogenic differentiation.7 Previous work developed biodegradable, phosphate-containing, methacrylated thermogelling macromers (MA-TGMs)8 that stabilize the hydrogels following thermogelation via chemical
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cross-linking. The macromers are made from a statistical copolymer of N-isopropyl acrylamide (NiPAAm), acrylamide (AAm), and monoacryloxyethyl phosphate (MAEP) that are esterified with glycidyl methacrylate (GMA) after polymerization, producing dual-gelling macromers that can form biodegradable hydrogels. These macromers have been shown to encapsulate cells with minimal cytotoxicity and undergo cell-induced mineralization in vitro.9 Furthermore, they have been shown to degrade and facilitate bone growth within a critical size rat-cranial defect.9 In an effort to improve cell-induced mineralization within the previously described hydrogels, HANP have been suspended in the macromer solutions prior to hydrogel formation to form hydrogel composites. Two formulations (10 and 13 mol% MAEP content) of these dual-gelling macromers were selected for forming hydrogel composites for physicochemical and in vitro characterization. The objective of the physicochemical studies was to evaluate the effect of suspended HANPs on the mechanical properties (as measured by compressive moduli) and the ability of the dual-gelling macromers to form stable hydrogels (as measured by swelling ratio). We hypothesized that for the HANP concentration tested (0.75 %w/v) the presence of HANPs would not interfere with stable gel formation (no change in swelling ratio) and that the incorporation of HANPs would not affect the compressive moduli of hydrogels. The objective of the in vitro studies was to evaluate the MSC-laden and acellular HANP hydrogel composites, synthesized from macromer solutions/HANP suspensions, for their ability to encapsulate rat MSCs and characterize cell viability, activity of the mineralization-facilitating enzyme alkaline phosphatase (ALP), and mineralization under osteogenic conditions.
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Methods Materials MAEP was purchased from Polysciences Inc. (Warrington, PA). NiPAAm, AAm, azobisisobutyronitrile (AIBN), GMA, dimethyl sulfoxide (DMSO), sodium phosphate dibasic, butylated hydroxytoluene (BHT), ammonium persulfate (APS), tetramethylethylenediamine (TEMED), acetic acid, β-glycerol 2-phosphate, dexamethasone, ampicillin, amphotericin, gentamicin and HANP (size
